The purpose of this project is to use the structure of the full length biotin-containing pyruvate carboxylase which we have recently solved to determine the details of the catalytic mechanism of this important metabolic enzyme. Our specific aims are: 1) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the biotin carboxylase domain where MgATP and bicarbonate carboxylate biotin. Further X-ray structures will be obtained of mutants and with bound reactants other than MgATP. 2) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the carboxytransferase domain where carboxybiotin converts pyruvate to oxaloacetate. Further X-ray structures will be obtained of mutants and with bound analogs of pyruvate. 3) To clarify the role of acetyl-CoA as an allosteric activator, X-ray crystal structures will be determined in its absence as well as its presence. Structures will be determined with enzymes from sources where acetyl-CoA is not an activator. Structures will also be determined in the presence of aspartate, an allosteric inhibitor, to determine where it binds and how it affects the structural changes caused by acetyl-CoA. The relationship between acetyl CoA binding and steps in the catalytic cycle with respect to the postulated half-of-the sites reactivity will be probed using fluorescent acetyl CoA analogues. 4) Investigation of the interdomain movement of biotin and carboxybiotin will be carried out using 1D and 2D NMR. [1-15N]-biotin and the methyl and acetyl analogs will be covalently attached to the enzyme using biotin ligase and a biotin auxotroph system. This label will allow us to probe the location and to determine to what extent the biotin is present in each domain. The kinetics of conformational changes associated with interdomain movements will be investigated by incorporation of a tryptophan and a coumaryl fluorescent amino acid analogue into specific sites in the BCCP and CT domains. The proximity of these residues, which varies according to the conformation of the pair of subunits, will be measured by fluorescence resonance energy transfer, using stopped-flow methods. 5) Carboxyphosphate will be synthesized by saturating a solution of tris[tetrabutylammonium] phosphate in dimethyl formamide with CO2. A small amount of this solution will be mixed in a stopped flow apparatus with enzyme, Mg2+, ADP and buffer and the resulting mixture analyzed for ATP formation with a firefly luciferase assay. If ATP is formed, this will establish carboxyphosphate as an intermediate in the reaction. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the structure and function of pyruvate carboxylase, an essential enzyme in the normal function of major organs such as liver, kidney, brain, pancreatic islets, mammary gland and adipose tissue. Deficiency leads to loss of control of normal blood sugar levels, severe and widespread metabolic disturbances, including abnormal brain function and early death, and overexpression is associated with type 2 diabetes and obesity. The tools used in the study are X-ray crystallography, creation of key mutants and their characterization by steady state and pre-steady state kinetics.